Opinion: Transactivation joins multiple tracks to the ERK/MAPK cascade

© 2003 Nature Publishing Group

PERSPECTIVES

The transactivation conceptIt has long been known that various stressfactors, such as ultraviolet (UV) and ioniz-ing radiation, can activate RTK signalling inthe absence of exogenously-added cognateligands4,5 (TABLE 1). It has since been foundthat the activation of GPCRs can, in turn,stimulate the signalling activity of RTKs(TABLES 1 and 2). This phenomenon wastermed RTK transactivation by Axel Ullrich’sgroup6, which discovered that several GPCRagonists had a stimulatory effect on the epi-dermal growth factor (EGF) receptor(reviewed in REFS 7–12). They initially foundthat in Rat-1 fibroblasts, endothelin-1,lysophosphatidic acid (LPA) and thrombininduce rapid phosphorylation of the EGFreceptor (EGFR). Suppression of EGFRtyrosine kinase activity by the specificinhibitor AG1478 strongly diminished theERK/MAPK activation that is mediated byGPCR agonists. This indicated an importantrole for EGFR activation in GPCR-mediatedstimulation of the ERK/MAPK cascade.Later, it was shown that EGFR transactiva-tion by GPCRs is a more general phenome-non that occurs in many cell types throughseveral very different GPCR species13 (TABLE 1).GPCR-mediated RTK transactivation mighthave a crucial role in diseases such as cardiachypertrophy14 and cancer15,16 and conse-quently has important implications for drugdevelopment. For example, owing to theirtransactivation effects, widely used GPCRagonists or antagonists might present a car-cinogenic risk or have beneficial effects inthe treatment of cancer, respectively.

Originally, it was believed that EGFRtransactivation by GPCR agonists occurs in aligand- (that is, EGF-) independent fashion.Again, Ullrich’s group showed that theGPCR-dependent stimulation of the EGFRmight involve stimulation of membrane-bound metalloproteinases, which, in turn,induce the extracellular release of heparin-binding-EGF (HB-EGF) from its latent,membrane-spanning precursor in theplasma membrane. So, one way in whichagonist binding to GPCRs might induceERK/MAPK activation is proposed to involveintracellular stimulation of a metallopro-teinase and extracellular shedding of HB-EGF from the plasma membrane, which, inturn, activates the EGFR and the mitogeniccascade. Data in support of this model havebeen obtained by several laboratories14,17,18

(TABLE 1). Shedding of HB-EGF or anotherEGFR ligand, such as transforming growthfactor-α (TGF-α), also seems to be involvedin EGFR activation by other factors, such asHelicobacter pylori19 or ionizing radiation20.

At present, this ‘triple-membrane-passing-signalling’ (TMPS)19,21 mechanism of GPCR-induced EGFR activation is a widely acceptedmodel of RTK transactivation. According tothis model, agonist-induced signals need topass further hurdles in the membrane beforethey are allowed to enter the intracellularspace. Intriguingly, as a functional equivalentto their ‘serpentine-like’ structure, GPCRsseem to induce serpentine-like signals (FIG. 2).

Certain aspects of the signalling pathwayfrom GPCRs to ERK/MAPK have not yetbeen well characterized. In particular, theidentity and intracellular control mecha-nisms of the metalloproteinases remain to beelucidated (TABLE 3).

Expansion of the concept Additional pathways of EGFR transactiva-tion that do not involve metalloproteinase-mediated HB-EGF release have also beenidentified. For example, the tyrosine kinasesSrc and Pyk have been proposed to mediateEGFR activation downstream of GPCR

Many agonists of G-protein-coupledreceptors (GPCRs) can stimulate receptortyrosine kinases and the extracellular signal-regulated kinase (ERK)/mitogen-activatedprotein kinase (MAPK) pathway. A ‘transactivation’ mechanism, which linksthese events in one signalling chain, inspiredmany researchers, but inevitably raised newquestions. A ‘multi-track’ model for GPCR signalling to the ERK/MAPK pathwaymight resolve some of the puzzles in thetransactivation field.

Several mitogenic signalling pathways thatconnect plasma-membrane receptors to vari-ous effector systems have been described. Oneparticular pathway that induces cell prolifera-tion through the activation of receptor tyro-sine kinases (RTKs) involves the signallingproteins Shc (Src-homology-2-containing),Grb2 (growth factor receptor-bound 2), Sos(son-of-sevenless), Ras, Raf, MEK (mitogen-activated protein kinase (MAPK) and extra-cellular signal-regulated kinase (ERK)kinase), and ERK/MAPK, and is known as theERK/MAPK cascade1 (FIG. 1a).

The G-protein-coupled receptor (GPCR)class comprises numerous members (FIG.1b; fora recent review, see REF. 2), and in various celltypes GPCRs can activate the ERK/MAPK cas-cade3 and induce cellular proliferation.However, the mechanism by which signals aretransduced from GPCRs to the ERK/MAPKcascade has been a matter of debate for manyyears.Recent advances indicate that RTK trans-activation is an important pathway that linksGPCRs and ERK/MAPK activation (BOX 1).

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 4 | AUGUST 2003 | 651

Transactivation joins multiple tracksto the ERK/MAPK cascade

Reinhard Wetzker and Frank-D. Böhmer

O P I N I O N


652 | AUGUST 2003 | VOLUME 4 www.nature.com/reviews/molcellbio

P E R S P E C T I V E S

receptor26, the neurotrophin receptors TrkAand TrkB27, and the fibroblast growth factor(FGF) receptor28 (TABLE 2).

A possible mechanism that could affectthe activity of many RTKs is the inactivationof protein-tyrosine phosphatases (PTPs).PTPs tightly control RTK activity and sig-nalling (for a review, see REF. 29). It is alsobelieved that transient, reversible PTP inacti-vation is involved in RTK signalgeneration1,30. Hydrogen peroxide (H

2O

2)

and other reactive oxygen species have beenshown to inactivate PTPs by oxidizing thecatalytic cysteine in the PTP active site. RTKactivation is also associated with H

2O

2gener-

ation, which presumably leads to thereversible inactivation of crucial PTPs31.Interestingly, the stimulation of many GPCRsalso leads to the generation of H

2O

2(see REF. 32

for a review), so GPCR-mediated H2O

2gen-

eration might inactivate the PTPs that nega-tively control RTKs and thereby lead to RTKtransactivation (FIG. 3b). Indeed, H

2O

2genera-

tion is essential for both LPA-stimulatedERK/MAPK activation in HeLa cells33 andthrombin-induced cell proliferation in vascu-lar smooth-muscle cells34. Technical difficul-ties in characterizing reversibly oxidized PTPsmight previously have precluded their directidentification as mediators of GPCR–RTKtransactivation. A recently introduced tech-nique to show reversible PTP inactivation30

might change this situation.Taken together, these results point to a

series of mechanisms that can potentiallyachieve signal transfer from GPCRs to RTKs.So, intracellular protein kinases, scaffold pro-teins and PTPs should be considered as medi-ators for this crosstalk in addition to, orinstead of, metalloproteinases and releasedgrowth factors.

that could be activated by proteolytic cleav-age. Although new, latent ligands of PDGFR PDGFC and PDGFD have recentlybeen discovered25, their possible participa-tion in a transactivation mechanism has notyet been investigated. There is also littleknown about the detailed mechanisms forthe GPCR transactivation of other RTKs,such as the insulin growth factor-1 (IGF-1)

activation (FIG. 3a). Both kinases have beenshown to interact with the EGFR22–24, andSrc has even been shown to directly phos-phorylate and activate the EGFR23.

An alternative mechanism to activate theEGFR might be through the formation of mul-timeric protein complexes and other forms offunctional compartmentation, such as assem-bly in membrane microdomains. β-arrestin,which is a facultative binding partner of theGPCRs, has been shown to engage in severalinteractions with signalling proteins such asSrc and other members of the MAPK cas-cades, and might function as a scaffold for theassembly of such multimeric complexes9. But,so far, little is known about the compartmen-tation of the EGFR and its possible role inmediating GPCR signalling. Such knowledgemight be important to gain a better under-standing of the transactivation concept.

Transactivation by GPCRs has also beenshown for other RTKs, such as the platelet-derived growth factor receptor (PDGFR)(TABLE 2). However, an interesting questionwould be the possible involvement of met-alloproteinases in this transactivationmechanism, and therefore the applicabilityof the TMPS model. The model wouldrequire the existence of latent growth factors

Figure 1 | Signal transduction of GPCRs and RTKs. a | Receptor tyrosine kinase (RTK) ligands bind tothe extracellular domain of RTKs. This leads to dimerization of two receptor subunits and intracellulartrans-autophosphorylation on tyrosine residues. Inhibition of receptor-directed protein-tyrosinephosphatases (PTPs) is another important aspect of receptor activation. Signalling molecules then bind tothe phosphotyrosine sites, which leads to their activation. One important signalling pathway is indicated:the adaptor molecule Grb2 binds directly or indirectly through another adaptor, Shc, and recruits Sos tothe receptor, which loads GTP onto Ras. Ras then activates the ERK/MAPK cascade — through Raf andMEK — which is linked to activation of cell proliferation. b | G-protein-coupled receptors (GPCRs) have awide range of relatively small, structurally diverse ligands. Extracellular ligand binding to GPCRs leads toGTP-loading of the Gα-subunit and dissociation of the trimeric G-protein complex from the receptor. Gα and Gβγ subunits elicit intracellular signals through protein–protein interactions. Many GPCRs can alsoactivate the conserved ERK/MAPK pathway and stimulate cell proliferation in some cell types. Active andinactive states are indicated by ‘traffic light’ coding — green and red, respectively. ERK, extracellular signal-regulated kinase; Grb2, growth factor receptor-bound 2; MAPK, mitogen-activated protein kinase; MEK,MAPK and ERK kinase; Shc, Src-homology-2-containing; Sos, son-of-sevenless; TM, transmembrane.

P PPPP

P

PPP

P

Grb2/SosShc

RasRaf

MEK

Proliferation

RTK(inactive)

a RTK(active)

GPCR(inactive)

b GPCR(active)

ERK/MAPKRasRaf

MEKERK/MAPK

Ligandbindingdomain

LigandLigand

TM domain 7TM(serpentine)structure

Kinasedomain

Proliferation

Other responses

PTP (active)

PTP (inactive)

α

α

γβ

γβ?

Plasmamembrane

Box 1 | Explanation of terms

Transactivation The activation of a given receptor activates a heterologous receptor. The latter can be of the sameor a different class with respect to the signal transmission mechanism. Aspects of signalling ofthe former receptor will be executed through the latter. This term is most frequently used in thecontext of receptor tyrosine kinase (RTK) activation by G-protein-coupled receptor (GPCR)agonists. It must be distinguished from transactivation of genes by transcription factors.

Triple-membrane-passing-signalling (TMPS) This is a new term for a particular type of transactivation that involves a sequence of threetransmembrane signalling events: GPCR activation; followed by membrane metalloproteinaseactivation; and subsequent activation of the epidermal growth factor receptor (EGFR) byheparin-binding EGF (HB-EGF) or other latent ligands of the EGFR. TMPS might be involved inthe activation of the EGFR by many heterologous effectors.

Transinactivation In this scenario, activation of a given receptor inactivates a heterologous receptor. Despitenumerous examples, so far this term is not frequently used.



seem to be dispensable for stimulation ofERK/MAPK by the GPCR agonists.

Together, these data indicate that, at least insome cellular systems,GPCR-dependent stimu-lation of the ERK/MAPK cascade does notinvolve RTKs. Additional signalling ‘tracks’seem to substitute for the TMPS signallingpathway through the plasma membrane.Transactivation seems to be important — butnot always essential — for the mitogenic sig-nalling of GPCRs. Following this idea, it couldbe proposed that some GPCRs stimulateERK/MAPK through RTK transactivation,whereas others use alternative pathways. Someexperimental findings, however, challenge thesimple supposition of one GPCR feeding intoone mitogenic signalling track. For example,LPA, an agonist for the EDG (endothelial dif-ferentiation gene) family of GPCRs, has beenshown to induce EGFR transactivation andstimulation of ERK/MAPK in many celltypes. Pertussis toxin, which is a specificinhibitor of G

isubunits of heterotrimeric

G proteins, either does not suppress8, or onlypartially suppresses13,61, increased phosphory-lation of the EGFR. However, pertussis toxinmostly blocks GPCR-induced stimulation ofERK/MAPK8,13,39.We believe that the simplestinterpretation of these data is a ‘dual-signalling-track’ model connecting LPA, Ras andERK/MAPK (FIG. 4). One track is mediated byG

i, and so is inhibitable by pertussis toxin,

whereas a second track, possibly throughanother G protein, feeds into the transactiva-tion pathway. Both RTKs, or alternative media-tors such as PKC,Rap1,B-Raf or PI3Kγ, conveysignals to ERK/MAPK. The relative contribu-tions of both parallel tracks might determinethe extent of pertussis toxin susceptibility.

Preliminary evidence for dual-track,or evenmulti-track, regulation of the ERK/MAPK cas-cade has also been obtained in several other cel-lular systems35.For example,Della Rocca et al.40

uncovered one tyrosine-kinase-independentand two tyrosine-kinase-dependent signallingpathways that are induced by agonists ofGPCRs in fibroblasts. The tyrosine-kinase-independent track is initiated by G

qsubunits of

heterotrimeric G proteins and mediated byPKC, whereas the tyrosine-kinase-dependentpathways involve either focal adhesion kinase(FAK) and/or EGFR transactivation. The rela-tive importance of these signalling tracks, how-ever, has subsequently been found to be highlycell-type specific. Similarly, in COS-7 cells,bradykinin type-2-receptor-mediated activa-tion of the ERK/MAPK cascade requires dual-track signalling through PKC and EGFRtransactivation41. In cardiac fibroblasts, stim-ulation of the ERK/MAPK cascade throughthe thrombin receptor/protease-activated

Multi-track signallingThere are a number of alternative mechanismsby which GPCR-mediated activation of theERK/MAPK cascade can take place. For exam-ple, a tyrosine-kinase-independent pathwaythat involves G

q-dependent activation of

phospholipase C (PLC)-β and protein kinaseC (PKC), and that subsequently phosphory-lates and stimulates c-Raf (the first member ofthe ERK/MAPK cascade), has been found in anumber of experimental systems35.Also, phos-phoinositide 3-kinase-γ (PI3Kγ) has beenidentified as another candidate for the directcoupling of GPCRs to the mitogenic cascade36.

The discovery of the transactivation pathwaystimulated many laboratories to test the partici-pation of EGFRs or other RTKs in their experi-mental systems.Several recent reports have pro-vided further examples of cases in which GPCRagonists might stimulate the ERK/MAPK cas-cade independently of RTK activation.

Schmitt and Stork analysed β2-adrenergic-

receptor-dependent stimulation of ERK/MAPKin human embryonic kidney 293 cells, but

addition of the EGFR inhibitor AG1478 didnot seem to have an effect on the stimulationof ERK/MAPK37. They proposed a signallingmechanism that involves the stimulatory G

s

subunit of heterotrimeric G proteins, cyclicAMP, protein kinase A, the small GTPase Rap1and the protein kinase B-Raf. This pathwaydoes not involve the metalloproteinase–HB-EGF–EGFR system. However, in contrast tothe absence of EGFR transactivation for theβ

2-adrenergic receptor,AG1478 clearly inhibits

the ERK/MAPK stimulation induced by theGPCR agonists thrombin and endothelin-1 inhuman embryonic kidney 293 cells21.

Schlessinger and co-workers recentlyapplied a genetic approach38 to analyse therole of the EGFR, Pyk2 and Src in signaltransduction from GPCRs to ERK/MAPK.They used fibroblasts that were deficient inSrc and Pyk2 to show that the LPA-dependentphosphorylation of the EGFR, which isinduced by the GPCR agonists LPA,bradykinin or carbachol, requires Src andPyk2. By contrast, Src, Pyk2 and the EGFR


Table 1 | EGFR activation by different agents*

Agent Cell type References

Stress factors

Ionizing radiation A431 cells 5,20

Oxidants EGFR-transfected B82 L-cellsHeLa cells 4Rat-1 cells

UV radiation EGFR-transfected B82 L-cells 4

G-protein-coupled receptor agonists

Angiotensin II Cardiomyocytes 14

ATP Primary mouse astrocytes 13

Bombesin Bombesin-receptor-transfected COS-7 13

Bradykinin PC12 rat pheochromocytoma cells 52Bradykinin type-2-receptor transfected COS-7 cells 41

Carbachol M1R- and M2R-transfected COS-7 13

Endothelin Rat-1 fibroblasts 6Cardiomyocytes 14

Lysophosphatidic acid Rat-1 fibroblasts 6Mouse embryo fibroblasts 38Squamous-cell carcinoma lines 15

Phenylephrine Cardiomyocytes 14

Thrombin Rat-1 fibroblasts 6Cardiac fibroblasts 42Cardiac myocytes 45

Other agents

Helicobacter pylori Gastric epithelial tumour cells 19

Integrin ligands Human primary skin fibroblastsECV304 endothelial cells 53

IGF-1 Mammary epithelial cells 51

Phorbol ester JB6 P+ (1-1) cellsMouse embryo fibroblasts 54

*Direct ligands of the EGFR are not included. EGFR, epidermal growth factor receptor; IGF-1, insulin-likegrowth factor 1; M1R and M2R, muscarinic receptors 1 and 2; UV, ultraviolet. An extended version of thistable can be found as a supplement to the online version of this article.




another GPCR, β-adrenergic receptor, in thesame cells. This leads to inhibition of the PKCtrack and abrogation of bradykinin-mediatedERK/MAPK activation in spite of maintainedEGFR transactivation. Multi-track signallingfrom the GPCR to ERK/MAPK might also beimportant in the regulation of a response overtime and in enabling sustained signalling. Forexample, a track through PKC is more impor-tant at early time points in thrombin activationof ERK/MAPK through protease-activatedreceptor-4 (PAR4) in cardiac myocytes,whereas EGFR activation is predominantlyrequired at later time points of stimulation45.

Basal activities and permissive signalsThe signalling tracks discussed so far are nor-mally induced by the addition of extracellularagonists to cellular systems. There is increas-ing evidence, however, that GPCRs, RTKs andsignalling molecules such as PI3K, Ras orERK/MAPK also show significant basal activ-ities in the absence of added agonists. As astriking example, Siekhaus and Drubinrecently described agonist- and receptor-independent signalling and the subsequentmating response that is induced by the basalactivity of heterotrimeric G proteins inyeast46. The following section illustrates thepossible regulatory significance of agonist-independent signalling activities and theirpotential importance for transactivation andadjoining signalling tracks.

Initial evidence for the regulatory effects ofthe basal activity of PI3K was presented byWennström and Downward47, who investi-gated the role of PI3Ks in the activation ofthe mitogenic cascade by EGF in fibroblasts.They found that stimulation of Ras andERK/MAPK by low but not high con-centrations of EGF was completely sup-pressed by PI3K inhibitors. Heterologouselevation of PI3K activity was, however, com-pletely ineffective at inducing Ras orERK/MAPK stimulation on its own. The

to control the ERK/MAPK module.Regulation of ERK/MAPK by two cooperat-ing signalling pathways might be a more gen-eral phenomenon and has, for example,recently been proposed to mediate greaterplasticity in B-cell-receptor-mediatedERK/MAPK activation43. This interpretationmight also apply to parallel signalling tracksthat are induced by GPCRs. For example,according to recent data44, dual-track sig-nalling of the bradykinin type-2 receptor toERK/MAPK through PKC or EGFR transacti-vation can be modulated by co-stimulation of

receptor-1 (PAR1) requires EGFR transactiva-tion, whereas EGFR signalling is not requiredfor PAR1-mediated stimulation of ERK/MAPKin cardiac myocytes42.Yart et al.39 investigatedLPA signalling in Vero kidney cells and foundtwo cooperating signalling pathways onethat is mediated by Gβγ and PI3Kβ, and anotherthat involves EGFR transactivation.Accordingto the data, both pathways are essential for fullERK/MAPK stimulation by LPA.

Together, these findings support the pro-posed multi-track concept. Distinct GPCRsseem to use more than one signalling pathway

Table 2 | Activation of other receptor tyrosine kinases by G-protein-coupled receptors

RTK Activation by Cell type Remarks References

FGFR1 Angiotensin II Bovine adrenal medulla cells Nuclear receptor 28action proposed

HER2/ErbB2/neu Lysophosphatidic acid SSC-9 squamous-cell carcinoma cells 15

IGF-1R Thrombin Vascular smoooth-muscle cells 26,55

Trk A,B Adenosine PC12 cells, hippocampal neurons Increased survival 27

PDGFR Lysophosphatidic acid L-cells 56Angiotensin Vascular smooth-muscle cells 57

VEGFR Bradykinin Primary cardiac capillary eNOS activation 50Endothelial cells downstream of VEGFR2

eNOS, endothelial nitric oxide synthase; FGFR1, fibroblast growth factor receptor 1; IGF-1R, insulin-like growth factor-1 receptor; PDGFR, platelet-derived growth factor;Trk, neurotrophin tyrosine kinase receptor; VEGFR2, vascular endothelial growth factor receptor 2.

Figure 2 | The ‘triple-membrane-passing-signalling’ model. G-protein-coupled receptor (GPCR)activation leads to the stimulation of different RTKs and the subsequent activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. The process is known asGPCR–receptor tyrosine kinase (RTK) transactivation and involves different mediators such as Src-familykinases, calcium (Ca2+), Pyk2 and protein kinase C (PKC), depending on the cell type. GPCR transactivation ofthe epidermal growth factor receptor (EGFR) occurs in many cell types through generation of a cognate ligand,the heparin-binding EGF (HB-EGF), which activates the EGFR and subsequently the ERK/MAPK cascade.HB-EGF is generated through extracellular proteolytic cleavage of proHB-EGF — a membrane-spanning,latent form of this growth factor — that is mediated by the action of a metalloproteinase (MP). A similaractivation might also occur for other latent growth factors, such as the precursor of transforming growth factor-α (proTGF-α). GPCR activation leads to metalloproteinase activation through several possiblemediators. As this pathway involves three signalling steps traversing the membrane, it has been designated‘triple-membrane-passing-signalling’ — TMPS — by the group of Axel Ullrich. Reminiscent of the structure ofGPCR, the signal passes the membrane in a ‘signalling serpentine’. MEK, MAPK and ERK kinase.

P P

P

P

PP

P

P

PP

RasRaf

MEKERK/MAPK

αγβ

Ca2+

Pyk2PKCSrc

MP (active)MP

Release ofHB-EGF (TGFα)

proHB-EGF(proTGFα)

GPCR ligand

GPCR (active)

1(outside–in)

3(outside–in)

2(inside–out)

EGFR (active)

Mediators



activity in the GPCR-dependent regulation ofthe ERK/MAPK cascade. At least in some celltypes, the agonist-independent fraction of theEGFR, or other RTK, activities could modu-late GPCR signal transduction to the mito-genic cascade.

Conclusion and perspectivesThe transactivation concept significantly con-tributes to the available knowledge on theinterplay of membrane-related signalling pro-teins. Possibly even more importantly, it isacting as a driving force for further experi-mental investigation.

Naturally, there are many unresolvedissues. The proposed regulatory effects ofGPCRs on metalloproteinases are mechanisti-cally ill-defined. Also, a precise qualitativecharacterization of the signalling eventsdownstream of the RTKs that have beentransactivated seems important in under-standing the specific features of the transacti-vation pathway. For example, when comparingEGFR signalling reactions that are inducedeither by EGF or by agonists of the β-adrenergicreceptor, Liebmann and co-workers recentlyfound significant differences. Whereas theaddition of (external) EGF strongly increasedPLCγ activity, agonists of the β-adrenergicreceptor did not induce the same effect,despite the occurrence of significant EGFRtransactivation as shown by RTK phospho-rylation (C. Liebmann, personal communi-cation). These data indicate differentialfunctions of EGFR ‘compartments’ or sub-species that are involved in direct activationby EGF- or GPCR-dependent transactivationmediated by HB-EGF. A further point thatrequires investigation is the possible activationof other latent growth factors by GPCR-depen-dent metalloproteinase activation — latent

authors concluded that the activation of themitogenic cascade by low concentrations ofgrowth factor requires basal PI3K activity. Theeffect of PI3K inhibitors on the activity of Rasor ERK/MAPK would therefore reflect a per-missive, rather than an upstream-regulatory,function of PI3K in this system47. Such a per-missive function of PI3K in the control of Rashas been confirmed in another report48, whichshowed that stimulation by phorbol esters canbe blocked by PI3K inhibitors in spite of theabsence of any effect of phorbol esters onPI3K activity.

Discussing recent data that seem tooppose the GPCR-dependent transactiva-tion of EGFR, Kranenburg and Moolenaarsuggested that basal EGFR tyrosine kinaseactivity might have a permissive role inGPCR-mediated ERK/MAPK activation8.Following this idea, it could be proposed that,at least in some cellular systems, the EGFR isnot directly involved in the transfer of theGPCR signal to Ras and ERK/MAPK, but thatit affects this signalling track as a feederthrough its agonist-independent basal activ-ity. Notably, this supposition allows the inter-pretation of the effects of pertussis toxin,which blocks GPCR-dependent ERK/MAPKstimulation in many cells but often fails toalter EGFR tyrosine kinase activity.

So, how can such a dual-track signallingmechanism be investigated? The bestoption seems a careful and quantitativeanalysis of the signalling characteristics ofthe EGFR, as has been performed for PI3Kin the reports mentioned above. The effectsof specific EGFR inhibitors on theautophosphorylation of the EGFR and onthe activities of Ras and ERK/MAPK in thepresence of different concentrations ofGPCR agonists should be investigated. Ifthe EGFR tyrosine kinase is essential forGPCR-dependent stimulation of Ras (andERK/MAPK) — as proposed by the trans-activation concept — then GPCR agonistsshould be incapable of affecting Ras and

ERK/MAPK activities following blockade ofthe EGFR. By contrast, a permissive functionof the EGFR would allow significant signaltransfer from GPCR to Ras and ERK/MAPK,even at strong inhibition of EGFR. Data, forexample, that were obtained by Piiper et al.49,support a permissive function of the EGFR.The effects of the EGFR inhibitor AG1478on EGFR autophosphorylation, and onRas/ERK/MAPK activities in the presence ofthe GPCR agonist cholecystokinin (CCK),were investigated49. Remarkably, at a concen-tration of AG1478 that completely blockedEGFR autophosphorylation, the Ras andERK/MAPK activities retained sensitivity toseveral-fold stimulation by CCK. Theseresults argue against a unidirectional signaltransfer from GPCR through the EGFR toRas and ERK/MAPK. To establish a permis-sive function of the EGFR, the reactivity ofRas and ERK/MAPK must be analysed in thepresence of different concentrations of theGPCR agonist.

Despite the obvious deficiency of data,these considerations might motivate investi-gation of a signalling function of EGFR basal


Table 3 | Activation of metalloproteinases by GPCRs and other agonists

Metalloproteinase Substrate Activation by Cell type References

ADAM17/TACE Growth-hormone- Phorbol ester Fibroblasts 58binding protein

ADAM10 proHB-EGF Bombesin, LPA COS-7 59

ADAM12 proHB-EGF Phenylephrine receptor Cardiomyocytes 14Angiotensin II receptorEndothelin receptor

ADAM10 proHB-EGF Platelet-activating- Epithelial cells 60factor receptor

ADAM, a disintegrin and metalloproteinase domain; EGF, epidermal growth factor; GPCR, G-protein-coupledreceptor; LPA, lysophosphatidic acid; proHB-EGF, pro-heparin-binding EGF; TACE, tumour necrosis factor-αconverting enzyme.

Figure 3 | Alternative mechanisms for GPCR–RTK transactivation. Several other possiblemechanisms for receptor tyrosine kinase (RTK) activation in the absence of a cognate ligand have beenproposed in the literature. a | One mechanism involves the G-protein-coupled receptor (GPCR)-triggeredrecruitment of RTKs in a complex with cytoplasmic tyrosine kinases (for example, Src and Pyk) and thesubsequent activation of the RTK. This might also occur in higher-order complexes that involve scaffoldsor membrane microdomains. b | Alternatively, GPCR activation might lead to the production of hydrogenperoxide (H2O2) through the activation of NADPH oxidases. Protein-tyrosine phosphatases (PTPs), whichnegatively control RTK activity and are very sensitive to oxidation, are transiently inactivated (Cys–SOH) byhydrogen peroxide. In turn, RTK signalling is activated62,63. Cys–SH, reduced cysteine.

P PPPP

P

PPP

P

a b

SrcPyk2

NADPHoxidase

P PPPP

P

PPP

PPTP (active)Cys–SH

PTP (inactive)Cys–SOH

αγβ

αγβ

SrcPyk2

Activation

H2O2

Inactivation

RTK (active)RTK (active) RTK (inactive)GPCRGPCR




3. Faure, M., Voyno-Yasenetskaya, T. A. & Bourne, H. R.cAMP and βγ subunits of heterotrimeric G proteinsstimulate the mitogen-activated protein kinase pathway inCOS-7 cells. J. Biol. Chem. 269, 7851–7854 (1994).

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situations. Further establishing the possibleinvolvement of RTK transactivation in car-diovascular regulation and dysfunction14,50

and in different types of cancer15,16,51 willuncover new insights into the respective dis-ease mechanisms, and might also result innew treatment opportunities. Inhibitors ofmetalloproteinases, HB-EGF-binding mole-cules and EGFR tyrosine kinase inhibitorshave already emerged as new drugs for themodulation of GPCR signalling. So, trackingdown further details of GPCR multi-tracksignalling will certainly be important fromthe medical perspective.

Note added in proofRecent work by Gschwind et al. demonstratedLPA- and carbachol-stimulated release ofamphiregulin, another EGFR ligand, throughtumour necrosis factor-α converting enzyme-dependent cleavage of pro-amphiregulin V(REF. 61). Recently resolved crystal structuresindicate that reversible oxidation of the cat-alytic cysteine residue of PTP results in for-mation of a sulphenyl-amide62,63.

Reinhard Wetzker and Frank-D. Böhmer are at the Institute for Molecular Cell Biology,

Jena University Hospital, Drackendorfer Strasse 1,D-07747 JENA, Germany.

Correspondence to R.W.e-mail: [email protected]

doi:10.1038/nrm1173

1. Schlessinger, J. Cell signaling by receptor tyrosinekinases. Cell 103, 211–225 (2000).

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PDGF isoforms are obvious candidates. Also,more work is required to study the effects ofGPCRs on PTPs. Although PTPs have beenestablished as important modulators of RTKsignalling and clearly mediate ligand-inde-pendent RTK activation by stress factors29,their possible role in GPCR–RTK transactiva-tion (FIG. 3b) has not been addressed in mostinvestigations so far. The proposal that activa-tion of many GPCRs leads to H

2O

2produc-

tion32, and the potential of reactive oxygenspecies to inactivate PTPs, might define ele-ments of a signalling track from GPCRs todifferent RTKs.

An attractive area of future research isdelineated by the interplay of the transactiva-tion pathway and of parallel, RTK-indepen-dent, signalling tracks. Careful investigationsof the signalling activities of all proteins thatare involved, as well as the use of geneticapproaches, are essential for rationalizing thecomplicated communication betweenGPCRs and the mitogenic cascade. Forexample, it seems desirable to analyse GPCRsignalling in cells with genetically or other-wise depleted signalling molecules. In thiscontext, the concept of a permissive role ofsignalling proteins and tracks that showbasal activities deserves attention. On theone hand, these studies will establish the dif-ferent molecular mechanisms — that is, thesignalling tracks — that cells can potentiallyuse. On the other hand, they will character-ize the different networking situations — theconcrete participation of certain signallingtracks in certain cell types or in pathological

Figure 4 | Activation of ERK/MAPK through parallel tracks. G-protein-coupled receptors (GPCRs)can also activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)pathway in the absence of receptor tyrosine kinase (RTK) activation. Mediators in this pathway can, for example, be phosphatidylinositide 3-kinase (PI3Kγ), which activates the cascade by an unknownmechanism involving Ras. Protein kinase C (PKC) isoforms can activate Raf by direct phosphorylation.Through Rap1 or B-Raf, GPCRs can also activate MAPK and ERK kinase (MEK). In many cell types,parallel pathways from GPCRs to ERK/MAPK activation exist, which include the RTK transactivationprocess. We designate this phenomenon, which is highly cell-type dependent, ‘multi-track signalling’.

P P

P

P

PP

P

P

PP

RasRaf

MEKERK/MAPK

PI3Kγ

Rap1/B-Raf

Mediators

Mediators

αγβ

Transactivation

GPCR ligand

GPCR (active) RTK (active)

PKC

Track 1

Track 2



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AcknowledgmentsWe thank several colleagues who made data available aheadof press. We gratefully acknowledge critical reading of thismanuscript by I. Rubio, C. Liebmann, A. Östman, S. Hsieh andA. Uecker. Work in the authors’ laboratories is supported bygrants from the Deutsche Forschungsgemeinschaft, HumanFrontiers in Science (to R.W.), the European Union (to R.W andF.D.B.) and Deutsche Krebshilfe (to F.D.B.).

Online links

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In contrast to the aberrant control ofproliferation, apoptosis, angiogenesisand lifespan, the cellular mechanismsthat cause local invasion and metastasisof tumour cells are still poorlyunderstood. New experimentalapproaches have identified different typesof epithelial-plasticity changes in tumourcells towards fibroblastoid phenotypes ascrucial events that occur duringmetastasis, and many molecules andsignalling pathways cooperate to triggerthese processes.

The mechanisms that oncogenes or tumour-suppressor genes use during malignant trans-formation are increasingly well understood1,2.However, the emphasis that surrounds crucialevents during tumour progression is chang-ing, as many molecular and epigeneticchanges seem to cooperate in alteringtumour-cell behaviour. These crucial eventsinclude altered responses/interactions oftumour cells to humoral and cellular environ-mental cues, such as paracrine and autocrinefactors, tumour stroma and immune cells2,3.However, molecular understanding of these

Diverse cellular and molecularmechanisms contribute to epithelialplasticity and metastasis

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